The field of the invention is synthetic nucleic acid sequences for improved amplification and expression in a host organism, and methods of creating them.
It has been a goal of biotechnology to promote the expression of cloned genes for analysis of gene structure and function and also for commercial-scale synthesis of desirable gene products. DNA cloning methods have enabled the genetic modification of bacteria and unicellular eukaryotes to produce heterologous gene products. In principle, the genes may originate from almost any source, including other bacteria, animal cells or plant cells. Although this expression of heterologous genes is a function of a variety of complex factors, maximizing the expression of cloned sequences has been under intense and rapid development. Plasmid and viral vectors have been developed in both prokaryotes and eukaryotes that enhance the level of expression of cloned genes. In some cases the vector itself contains the regulatory elements controlling the expression of genes which are not normally expressed in the host cell so that a high level of expression of heterologous genes can be obtained.
Several problems exist, however, in the expression of many proteins across phyla and even across species. Post-translational handling and modification of expressed proteins by the host cell often does not mimic that of the heterologous gene""s own cell type. Frequently, even if the protein is expressed in a useful form, heterologous genes are poorly expressed. Low yields of expressed protein may make manufacture of commercially useful quantities impossible or prohibitively costly. Vectors designed to enhance expression are not able to overcome some expression problems if the regulatory elements of the vector are not the constraint on robust expression. Other cellular or translational constraints are at issue.
Genes encoding poorly expressed proteins are often themselves difficult to clone and amplify as well. This can be due to secondary structure inherent in the gene, for example caused by high G-C content. Some methods have been used to reduce these difficulties, such as the use of DMSO or betaine to bring G-C and A-T melting behaviors more into alignment, or the use of ammonium sulfate (hydrogen binding cations) to destabilize G-C bonding during PCR. The problem with these methods is that the effects of the additives are concentration dependent, so variations in template size and G-C content mean lengthy optimization procedures. Additionally, these steps do nothing to facilitate subsequent expression of the nucleic acid once it has been cloned.
The frequency of particular codon usage in Escherichia coli and other enteric bacteria has long been known, and it has been hypothesized that replacement of certain rare codons encoding a particular amino acid in a heterologous eukaryotic or prokaryotic gene with a codon that is more commonly used by the selected host bacterium (or eukaryotic host cell) would enhance expression (see, e.g., Kane, Curr Opin Biotechnol 6:494-500 (1995) and Zahn, J Bacteriol., 178:2926-2933 (1996)). This is based on the theory that rare codons have only a few tRNAs per cell and that transcription of heterologous sequences having numerous occurrences of these rare codons is limited by too few available tRNAs for those codons. However, simple replacement of rare codons does not reliably improve expression of heterologous genes, and no broadly applicable method exists to select which codon changes are best to increase expression of heterologous sequences. Further, it is not known in detail how codon usage is related to expression level.
Many gene products, often from bacteria, are commonly used as research and assay reagents, and various microbial enzymes increasingly are finding applications as industrial catalysts (see, for example, Rozzell, J. D., xe2x80x9cCommercial Scale Biocatalysis: Myths and Realities,xe2x80x9d Bioorganic and Medicinal Chemistry, 7:2253-2261 (1999), herein incorporated by reference). Some have substantial commercial value. Examples include heat-stable Taq polymerase from Thermus aquaticus, restriction enzymes such as Eco RI from E. coli, lipase from Pseudomonas cepacia, xcex2-amylase from Bacillus sp., penicillin amidase from E. coli and Bacillus sp., glucose isomerase from the genus Streptomyces, and dehalogenase from Pseudomonas putida. Genes from bacteria may express easily in commercially useful host strains, but many do not. In particular, genes from many bacteria have significantly different codon preferences from enteric bacteria. For example, filamentous bacteria such as streptomycetes and various strains of the genus Bacillus, Pseudomonas, and the like can be difficult to express abundantly in enteric bacteria such as E. coli. An example of a Pseudomonas gene that is difficult to express in E. coli is the enzyme methionine gamma-lyase, useful for the assay of L-homocysteine and/or L-methionine as described in U.S. Pat. No. 5,885,767 (herein incorporated by reference). This assay is particularly useful in the diagnosis and treatment of homocystinuria, a serious genetic disorder characterized by an accumulation of elevated levels of L-homocysteine, L-methionine and metabolites of L-homocysteine in the blood and urine. Homocystinuria is more fully described in Mudd et al., xe2x80x9cDisorders of transsulfuration,xe2x80x9d In: Scriver et al., eds., The Metabolic and Molecular Basis of Inherited Disease, McGraw-Hill Co., New York, 7th Edition, 1995, pp. 1279-1327 (herein incorporated by reference). In developing an assay for the accurate quantitation of L-homocysteine and L-methionine according to the methods described in U.S. Pat. No. 5,885,767, obtaining large amounts of methionine gamma-lyase is necessary. However, this Pseudomonas gene contains a number of codons that are less commonly found in genes of desirable bacterial hosts for expression such as E. coli. 
Similarly, genes from other organisms, such as yeast or mammals, can have utility as therapeutic agents, reagents, or catalysts. Examples include erythropoietin, human growth hormone, and eukaryotic oxidoreductases such as amino acid dehydrogenases, disulfide reductases, and alcohol dehydrogenases.
Because plasmid vectors designed to enhance expression with a variety of promotors or other regulatory elements often do not resolve the difficulty in expressing certain genes, and because no systematic approach exists for codon replacement to aid amplification of nucleic acids or their expression, there is clearly a need for an improved method for amplification and expression of genes, including genes from mammals and other animals, plants, yeast, fungi, and various bacteria such as streptomycetes, Bacillus, Pseudomonas and the like introduced into enteric bacterial hosts such as E. coli. 
In one embodiment, the invention is directed to a method of making a synthetic nucleic acid sequence. The method comprises providing a starting nucleic acid sequence, which optionally encodes an amino acid sequence, and determining the predicted xcex94Gfolding of the sequence. The starting nucleic acid sequence can be a naturally occurring sequence or a non-naturally occurring sequence. The starting nucleic acid sequence is modified by replacing at least one codon from the starting nucleic acid sequence with a different corresponding codon to provide a modified nucleic acid sequence. As used herein, xe2x80x9ccodonxe2x80x9d generally refers to a nucleotide triplet which codes for an amino acid or translational signal (e.g., a stop codon), but can also mean a nucleotide triplet which does not encode an amino acid, as would be the case if the synthetic or modified nucleic acid sequence does not encode a protein (e.g., upstream regulatory elements, signaling sequences such as promotors, etc.). As used herein, a xe2x80x9cdifferent corresponding codonxe2x80x9d refers to a codon which does not have the identical nucleotide sequence, but which encodes the identical amino acid. The predicted xcex94Gfolding of the modified nucleic acid sequence is determined and compared with the xcex94Gfolding of the starting nucleic acid sequence. In accordance with the invention, the predicted xcex94Gfolding of the starting nucleic acid sequence can be determined before or after the modified starting nucleic acid is provided.
Thereafter, it is determined whether the xcex94Gfolding of the modified nucleic acid sequence is increased relative to the xcex94Gfolding of the starting nucleic acid sequence by a desired amount, such as at least about 2%, at least about 10%, at least about 20%, at least about 30%, or at least about 40%. If the xcex94Gfolding of the modified nucleic acid sequence is not increased by the desired amount, the modified nucleic acid sequence is further modified by replacing at least one codon from the modified nucleic acid sequence with a different corresponding codon to provide a different modified nucleic acid sequence. These steps are repeated until the xcex94Gfolding of the modified nucleic acid sequence is increased by the desired amount to ultimately provide a final nucleic acid sequence, which is the desired nucleic acid sequence.
In one embodiment, the invention is a synthetic polynucleotide designed by the methods of the invention. This includes a nucleic acid having the sequence of a polynucleotide designed by the methods or a sequence complementary thereto.
In another embodiment, the invention includes a method of physically creating a tangible synthetic polynucleotide comprising creating a physical embodiment of the synthetic polynucleotide made using the nucleic acid/polynucleotide design methods of the invention, and the physical embodiments of the tangible synthetic polynucleotide prepared by this method (i.e., physical embodiments of the synthetic sequences, and copies of such sequences created by other methods.
The modified and/or final nucleic acid sequence can then be physically created. By the present invention, a desired nucleic acid sequence can be created that is more highly expressed in a selected host, such as E. coli, an insect cell, yeast, or a mammalian cell, than the starting sequence. By xe2x80x9cmore highly expressedxe2x80x9d is meant more protein product is produced by the same host than would be with the starting sequence, preferably at least 5% more, more preferably at least 10% more, and most preferably at least 20% more.
Preferably the codon replacement is in a region of the starting nucleic acid sequence or modified nucleic acid sequence containing secondary structure. It is also preferred that the different corresponding codon is one that occurs with higher frequency in the selected host. In a particularly preferred embodiment, the desired amino acid sequence is expressed in Escherichia coli, and the amino acid sequence is from a bacterium of the genus Pseudomonas, and the different corresponding codon is selected to be one that occurs with higher frequency in a selected host, such as Escherichia coli than does the replaced codon. Alternatively, or in addition, the different corresponding codon is selected as one that has fewer guanine or cytosine residues than the replaced codon.
In a particularly preferred embodiment, the starting nucleic acid sequence is derived, e.g., converted, from an amino acid sequence native to an organism different from the desired host for expression, for example Pseudomonas. 
The method of the invention also provides a modified, final sequence that is more amplifiable than the starting sequence. In other words, the final sequence is amplified more readily in a full length form, more rapidly or in greater quantity.
In another embodiment, the invention is directed to a synthetic nucleic acid sequence having a plurality of codons and encoding a methionine gamma-lyase protein from Pseudomonas putida. As used herein, the phrase xe2x80x9cnucleic acid sequence encoding a proteinxe2x80x9d means that the nucleic acid sequence encodes at least the functional domain of the protein. The sequence having no more than about 95% homology, preferably no more than about 90% homology, more preferably no more than about 85% homology, still more preferably no more than about 80% homology, to a naturally occurring methionine gamma-lyase gene from Pseudomonas putida. At least about 5%, preferably at least about 10%, more preferably at least about 20%, still more preferably at least about 30%, even more preferably at least about 40%, of the codons in the synthetic nucleic acid sequence are different from codons found in the naturally occurring gene.
In one aspect, the codons in the synthetic nucleic acid sequence encode the same amino acids as the codons in the naturally occurring gene. In another aspect, at least one of the codons in the synthetic nucleic acid sequence encodes an amino acid different from the numerically corresponding amino acid found in the naturally occurring sequence. In yet another aspect, at least one of the different codons in the synthetic nucleic acid sequence is in an area of secondary structure in the naturally occurring gene.
In another embodiment, the invention is directed to synthetic genes derived from any source, e.g., eukaryotic or prokaryotic, for improved expression in heterologous or homologous expression hosts. The synthetic nucleic acid sequences of the invention are comprised on non-naturally occurring polymers of nucleic acids, each sequence having a biological function encoded by the sequence. The biological function can be direct (e.g., the nucleic acid sequence possesses the function, as in a promotor, for example) or indirect (e.g., the nucleic acid serves as a template to encode another molecule such as RNA or protein which has a function), and is generally one that is known from a similar naturally occurring or synthetic sequence. However, the biological function of the synthetic sequence created using the methods of the invention need not be identical to a known or predicted biological function in a known starting sequence. For example, the function may be enhanced in the synthetic sequence, or an enzyme may act on one or more different substrates, use more or different co-factors, catalyze reactions at a different rate, etc. The synthetic sequences further have no more than about 95% homology to a known starting sequence, and have a different free energy of folding than does the starting sequence. Finally, the synthetic sequences of the invention have the characteristic that they are better expressed (e.g., more highly expressed, expressed under different conditions, or expressed with more desired characteristics) in a selected host cell than the starting sequence would be if expressed in the selected host cell. The host cell is generally heterologous, but may be homologous for the starting sequence (the artificial synthetic sequence, not being found in nature, has no homologous host).
In one aspect, the synthetic nucleic acid sequence comprises a plurality of codons which encode amino acids and proteins. In preferred embodiments, the difference between the synthetic sequence and the starting sequence is that the synthetic sequence has at least one codon which is different from the starting sequence at the same amino acid position in the protein sequence. This codon may encode a different amino acid, the same amino acid, insert or delete an amino acid from that position, or encode a restriction site. Members of the oxidoreductase family are disclosed, and all members of this family or sequences encoding oxidoreductase functionality are among preferred sequences. Other preferred sequences include those encoding decarboxylase, formate dehydrogenase, hydantoinase, and vanillyl alcohol oxidase functions. Any sequence encoding a biological function from any source can be improved using the methods of the invention for enhanced expression or functionality.
In another embodiment, the invention is directed to a method of creating a synthetic nucleic acid. The method comprises providing a sense nucleic acid sequence having a 5xe2x80x2 end and a 3xe2x80x2 end and providing an antisense nucleic acid sequence having a 5xe2x80x2 end and a 3xe2x80x2 end. Preferably the sense and antisense nucleic acid sequences are between about 10 and about 200 bases, more preferably between about 80 and about 120 bases. The 3xe2x80x2 end of the sense sequence has a plurality of bases complimentary to a plurality of bases of the 3xe2x80x2 end of the antisense sequence, thereby forming an area of overlap. Preferably the area of overlap is at least 6 bases, more preferably at least 10 bases, still more preferably at least 15 bases. The 5xe2x80x2 end of the sense sequence extends beyond the 3xe2x80x2 end of the antisense sequence, and the 5xe2x80x2 end of the antisense sequence extends beyond the 3xe2x80x2 end of the sense sequence. The method further comprises annealing the sense and antisense sequences at the area of overlap. A polymerase and free nucleotides are added to the sequences. Said nucleotides may be naturally occurring, i.e., A, T, C, G, or U, or they may be non-natural, e.g., iso-cytosine, iso-guanine, xanthine, and the like. The sequences can be annealed before or after addition of the polymerase and free nucleotides. The sequences are extended, wherein the area of overlap serves to prime the extension of the sense and antisense sequences in the 3xe2x80x2 direction, forming a double stranded product. The extended sequence can then be amplified. Further, a second step to the method can be added where the double stranded first extension product is separated into an extended sense strand and an extended antisense strand and a second set of sense and antisense nucleic acid sequences are provided having a 5xe2x80x2 end and a 3xe2x80x2 end. Each has a plurality of bases on its 3xe2x80x2 end complementary to a plurality of bases on the 3xe2x80x2 end of the extended sense or antisense strand respectively, thereby forming second and third areas of overlap. A polymerase and free nucleotides are added to the sequences and separated strands, wherein the second and third areas of overlap serve to prime a second extension of the sequences and strands that encompasses the sequence of the first sense and antisense nucleic acid sequences and the second sense and antisense nucleic acid sequences.